Whole-exome sequencing identification of a recurrent CRYBB2 variant in a four-generation Chinese family with congenital nuclear cataracts

Abstract

Congenital cataracts is the most common cause of visual impairment and blindness in children. Although there have been extensive studies into the pathogenesis of congenital cataracts, the pathogenic mechanism underlying the recurrent variant CRYBB2:c.62T>A(p.I21N) has not been previously reported. Thus, the present study aimed to use whole-exome sequencing (WES) to identify potential genetic variants and investigate how they may have induced the occurrence of cataracts in a four-generation Chinese family with congenital nuclear cataracts. The medical history of this family was recorded and WES was conducted for one proband. Sanger sequencing was used to verify the presence of the putative variant in all participants. PolyPhen-2, SIFT and ProtScale were used to analyze the effect of the identified variants on protein function and hydrophobicity, and Pymol was used to show the structure of the wild-type (Wt) and mutant β-crystallin B2 (CRYBB2) protein. Full-length Wt-CRYBB2 or mutant-CRYBB2 (I21N-CRYBB2) were fused to green fluorescent protein (GFP), and the recombinant plasmids were transfected into HeLa cells. Reverse transcription-quantitative PCR and western blotting were used to detect the expression levels of CRYBB2 mRNA and protein. Immunofluorescence and flow cytometry analyses were used to detect protein localization and apoptosis, respectively. A recurrent variant CRYBB2:c.62T>A(p.I21N) was identified in a four-generation Chinese family with congenital nuclear cataracts. Multiple-sequence alignment of CRYBB2 demonstrated that codon 21 was highly conserved. Pymol revealed that the structure of the I21N-CRYBB2 protein was distinct from that of Wt-CRYBB2. PolyPhen-2 predicted that it had a variant provean score 1.0, suggesting it was 'probably damaging', and SIFT predicted it had a variant provean score of -5.113, indicating it was 'deleterious'. ProtScale indicated that the hydrophobicity of the mutation site was significantly reduced. The protein expression levels of the I21N-CRYBB2 were decreased compared with the Wt-CRYBB2. Immunofluorescence analysis revealed that the variant I21N-CRYBB2 protein tended to accumulate around the nucleus, and flow cytometry analysis indicated that it increased cell apoptosis. Furthermore, I21N-CRYBB2 induced the activation of the unfolded protein response (UPR). In conclusion, a pathogenic variant of CRYBB2:c.62T>A(p.I21N) was identified via WES in a four-generation Chinese family with congenital nuclear cataracts. Through biological analysis, it was found that the variant induced abnormal protein aggregation, activated the UPR and triggered excessive cell apoptosis, which may lead to the occurrence of congenital nuclear cataracts in this family.

Keywords: CRYBB2; WES; autosomal dominant; cataract genetics; congenital cataracts; mutation; variant.

Figure 1

Family pedigree and cataract phenotype of proband. (A) Pedigree of the family. The squares and circles represent men and women, respectively. Solid and hollow indicate wild-type and variant individuals, respectively. The solid black arrow indicates the proband (IV.1), and the diagonal line indicates the deceased family member. (B) Image of the lens of the proband (IV.1).

Figure 2

Sanger sequencing and multiple-sequence alignment. (A) Sanger sequencing showed that CRYBB2:c.62T>A (p.I21N) was detected in all variant individuals. (B) Multiple-sequence alignment showed the variant p. I21N was located within a highly conserved region. CRYBB2, β-crystallin B2.

Figure 3

Structure of the CRYBB2 protein as predicted by Pymol. Green and red indicate the Wt and the variant, respectively. Pymol showed that a β-strand and an α-helix were altered in the variant protein and that the Wt and variant protein were not fully composite. CRYBB2, β-crystallin B2; Wt, wild-type; Mut, mutant.

Figure 4

PolyPhen-2 evaluation and hydropathy analysis. (A) Score obtained from PolyPhen-2 analysis was 1.0, indicating that the p. I21N variant was predicted to damage the structure and function of the CRYBB2 protein. (B) Analysis of hydropathy indicated that the hydrophobicity of the variant site was lower than that of the Wt. Hydrophobicity analysis of (a) Wt and (b) Mut. Wt, wild-type; CRYBB2, β-crystallin B2; Mut, mutant.

Figure 5

Expression levels of Wt and Mut-CRYBB2. (A) Wt-CRYBB2 and Mut-CRYBB2 were successfully expressed in transfected cells. Values are presented as the mean ± SD. One-way ANOVA and Bonferroni multiple comparison was used to test the differences between three groups, ^***P<0.001. (B) Mut-CRYBB2 protein expression levels were lower than those of Wt-CRYBB2 in transfected cells. GAPDH was used as the loading control. (C) Confocal laser scanning images showed that the Mut-CRYBB2 protein tended to surround the nucleus. Scale bar, 5 µm. CRYBB2, β-crystallin B2; Wt, wild-type; Mut, mutant; ns, no significance; GFP, green fluorescent protein.

Figure 6

Hoechst 33342 staining and flow cytometry analysis. (A) Hoechst 33342 staining showed increased apoptosis of cells transfected with the variant CRYBB2. The white arrow indicates an apoptotic cell. Magnification, x200. (B) Flow cytometry analysis demonstrated increased apoptosis in cells transfected with the variant CRYBB2. CRYBB2, β-crystallin B2; Wt, wild-type.

Figure 7

UPR-associated gene expression status in cells. (A) Western blot analysis confirmed the increase in BiP expression in cells expressing the Mut-CRYBB2. GAPDH was used as the loading control. Relative mRNA expression levels of (B) DDIT3, (C) HSPA5 and (D) IRE1. Values are presented as the mean ± SD. One way ANOVA and Bonferroni multiple comparison was used to test the differences between three groups. ^**P<0.01. (E) Xbp1 splicing detection (1, Vector; 2, cells expressing Wt-CRYBB2; 3, cells expressing Mut-CRYBB2). BiP, binding immunoglobulin protein; DDIT3, DNA damage-inducible transcript 3; Ire1, inositol-requiring enzyme 1; Xbp1, X-box-binding protein 1; HSPA5, heat shock protein family A (Hsp70) member 5; ns, not significance; Wt, wild-type; Mut, mutant.